Quantitative assay for human terminal complement cascade activation

ABSTRACT

The present invention discloses an enzyme-linked immunosorbent assay (ELISA) to quantitate fluid phase terminal complement activation. Upon activation to form C5b-9, terminal complement components express neoantigens not present in the unassembled individual components. Rabbit antiserum to polymerized C9 was rendered specific for C9 neoantigenic determinants by serial immunosorbtion with human serum, human C9, and other terminal complement components bound to Sepharose. Using the IgG from this antiserum, a sandwich ELISA was devised to bind SC5b-9 from solution onto polystyrene plates. The ELISA plates were developed with the use of goat antiserum to native C9 epitopes followed by a swine anti-goat IgG alkaline phosphatase conjugate. Quantitation of SC5b-9 in solution was performed by comparing sample OD to a standard curve generated with human SC5b-9 that was purified from zymosan-activated serum. The assay is sensitive to as little as 100 ng of SC5b-9/ml and is useful for screening plasma, serum, cerebrospinal fluid, or other biological fluids for the presence of terminal complement cascade activation.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention is related to a method of quantitation ofactivation of the human terminal complement cascade. More particularly,the present invention is related to a kit and a quantitative method forfluid phase human terminal complement cascade activation byenzyme-linked immunosorbent assay.

2. State of the Art

It has been reported that the terminal complement cascade is activatedin a variety of human diseases, including the glomerulonephritis andcutaneous lesions of systemic lupus erythematosus (Biesecker et al. J.Exp. Med. 154: 1779, 1981; Biesecker et al., N. Engl. J. Med. 306: 264,1982; Falk et al. Clin. Research 32:503A (Abstract), 1984), otherglomerulonephritides (Falk et al. J. Clin. Invest. 72:560, 1983),bullous pemphigoid (Dahl et al. J. Invest. Dermatol. 82:132, 1984),dermatitis herpetiformis (Dahl et al. Arch. Dermatol. 121:70, 1985), anddemyelinating diseases (Sanders et al. Clin. Research 33:388A(abstract), 1985). However, a quantitative and sensitive enzyme linkedimmunosorbent assay to determine or detect the activation of theterminal cascade in the human body fluid is not available. Therefore,the availability of a simple, sensitive assay which measures activationof the terminal complement components should prove useful for studyingthe pathophysiology of a variety of disease states and for monitoringdisease activity.

Activation of the terminal portion of the complement cascade results inthe assembly of a macromolecular complex consisting of C5b, C6, C7, C8and a variable number of C9 monomers. If activation occurs in the fluidphase, two or three molecules of S protein combine with the complex,making it cytolytically inactive (Kolb and Muller-Eberhard Proc. Natl.Acad. Sci. U.S.A. 72:1687, 1975). However, this SC5b-9 complex is watersoluble and contains 2-3 C9 monomers. In the presence of a targetmembrane, the activation of the terminal complement cascade results inthe formation of the MC5b-9 complex, which is cytolytically active(Mayer Proc. Natl. Acad. Sci. 69:2954, 1972). The MC5b-9 complexcontains a variable number of C9 monomers, as many as 12 to 16,depending on the availability of C9 (Podack et al. J. Exp. Med. 156:268,1982; Bhakdi and Tranum-Jensen J. Immunol. 133:1453, 1984). Furthermore,purified human C9 alone if incubated at 37° C. spontaneously formsclosed-ring, SDS-resistant polymerized C9 (poly C9) (Podack and TschoppProc. Natl. Acad. Sci. U.S.A. 79:574, 1982) and polymerization ofpurified C9 results in expression of a C9 neoantigen. This C9neoantigen- is also expressed in SC5b-9 and MC5b-9 complexes (Podack andTschopp J. Biol. Chem. 257:15204, 1982; Falk et al. J. Clin Invest.72:560, 1983), as are several other neoantigens related to other stepsin C5b-9 assembly (Kolb and Muller-Eberhard J. Exp. Med. 141:734, 1975).

Polyclonal antibodies directed to all of the neoantigens of SC5b-9 havebeen used in an immunoradiometric assay for SC5b-9 (Bhakdi and Muhly J.Immunol. Methods 57:283, 1983). This assay, which is based on theinhibition of binding of radiolabeled antineoantigen antibodies torabbit erythrocyte membranes bearing MC5b-9 lesions, is sensitive to 3to 4 μg of SC5b-9 per ml, equivalent to a 1% activation of the terminalcomplement components in normal serum. In another competitive inhibitionradioimmunoassay for SC5b-9 (Falk et al. Clin. Research 32:503A(abstract), 1984), a monoclonal antibody to the C9 neoantigen was used.In this assay, radiolabeled polymerized C9 was displaced from themonoclonal antibody by the SC5b-9 present in the test sample. A standardcurve was created using unlabeled poly C9, and the results wereexpressed as unit equivalents of poly C9 rather than as units of SC5b-9.The sensitivity of the assay was not reported. Finally, an enzyme-linkedimmunosorbent assay (ELISA) for SC5b-9 has been described which, in asandwich fashion, uses antibodies to native epitopes in two differentcomplement components present in the assembled C5b-9 complex. Althoughthis approach yields a sensitive assay for SC5b-9, the utility of theassay is limited in that its signal can be inhibited by normal humanserum (Mollnes et al. Scand. J. Immunol. 20:157, 1984).

The present invention discloses a method which does not have thelimitations or disadvantages of the prior art assays. Definitions ofcertain terms used herein may be found in Podack et al, Mol. Immunol.21:589-603, 1984 which is incorporated herein by reference.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to provide asimple, sensitive and quantitative assay to detect the activation ofterminal complement cascade.

It is a further object of the present invention to provide anenzyme-linked immunosorbent assay (ELISA) for quantitative determinationof terminal complement cascade activation using the human body fluid.

It is a still further object of the present invention to provide anELISA for C9 neoantigen which allows quantitation of about 100 ng orless of SC5b-9 per ml of the body fluid.

An additional object of the present invention is to provide a kitcomprising containers containing necessary components of the ELISA ofthe present invention.

Other objects and advantages of the present invention will becomeevident as the detailed description of the present invention proceeds.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and many of the attendant advantagesof the invention will be better understood upon a reading of thefollowing detailed description when considered in connection with theaccompanying drawings wherein:

FIG. 1 shows Coomassie-stained SDS-PAGE of SC5b-9, 6.5% acrylamide,non-reduced. There were bands of appropriate M_(r) to represent C5, C6,C7, C8α-γ, C8β, C9 and S protein. Minimal contamination with otherproteins was detected. The presence of C5, C6, C7, C8, and C9 was shownin this material by double diffusion and immunoelectrophoresis;

FIG. 2 shows ELISA for SC5b-9. Polystyrene plates were precoated withrabbit IgG specific for C9 neoantigen. After incubation with samples orSC5b-9 standards, the plates were developed with goat anti-C9 followedby swine anti-goat IgG alkaline phosphatase conjugate. A quantitativecolor reaction developed after the addition of P-nitrophenyl phosphatesubstrate;

FIG. 3 shows double diffusion in 1% agarose. Center well containedunabsorbed anti-C9 neoantigen. Precipitin lines are seen with poly C9 at12 and 6 o'clock and with MC5b-9 at 10 o'clock. Lines of partialidentify are seen with native C9 and 2 and 8 o'clock. No lines weredetected against normal plasma at 4 o'clock;

FIG. 4 shows quantitative ELISA run with dilution of purified SC5b-9.Points represent mean ±SD. A dose-response relationship with OD isdemonstrated;

FIG. 5 shows sensitivity level of quantitative ELISA for SC5b-9. TheELISA shows a curvilinear dose response to as little as 100 ng ofSC5b-9/ml;

FIG. 6 shows quantitative ELISA run with dilutions of purified poly C9and MC5b-9. Points represent mean ±SD. Dose-response relationships to ODare demonstrated for both poly C9 and MC5b-9; and

FIG. 7 shows quantitative ELISA run with dilution of normal humanplasma. Points represent mean ±SD. No dose-response relationship isdemonstrated.

DETAILED DESCRIPTION OF THE INVENTION

The above objects and advantages of the present invention are achievedby a method for assaying activation of terminal complement cascade,comprising in sequence the steps of:

(a) coating a suitable surface with a sufficient amount of a firstantibody having specificity for poly C9 antigen;

(b) washing said surface with a suitable medium to remove unboundantibody;

(c) incubating said surface with a sample to be assayed for sufficienttime so that binding reaction between said sample and the first antibodyis substantially complete;

(d) washing said surface with a suitable medium to remove unboundsample;

(e) incubating said surface with a second antibody specific for aconstituent of the terminal cascade for sufficient time so that bindingbetween antigen and the second antibody is substantially complete;

(f) thereafter washing the surface with a suitable medium to removeunbound second antibody;

(g) incubating said surface with a third antibody which recognizes thesecond antibody and which is conjugated with a suitable enzyme for asufficient time so that the binding between said second and thirdantibodies is substantially complete;

(h) washing said surface with a suitable medium to remove unbound thirdantibody;

(i) incubating the surface with a substrate specific for the conjugatedenzyme for sufficient time so that the enzyme-substrate reaction issubstantially complete; and

(j) measuring the product resulting from enzyme-substrate reactionrelative to known amounts of SC5b-9 standard similarly treated as insteps (a) thru (j).

The term "substantially complete" as used herein means that the reactionis as complete as can be expected under the test conditions.

The term "body fluid" as used herein means blood, plasma, serum,cerebrospinal fluid, urine and the like.

Although any similar or equivalent materials and methods can be employedfor the practice of the present invention, the following are thepreferred methods and materials. All publications cited hereunder areincorporated herein by reference.

Buffers

The following buffers were used: isotonic Veronal-buffered salineprepared with 0.1% gelatin, 0.15 mM CaCl₂, 1 mM MgCl₂, 0.15M NaCl, 1 g/ldiethyl barbiturate, pH 7.35 (GVBS⁺⁺); lysine-Sepharose buffercontaining 0.15M NaCl, 0.01M ethylenediaminetetraacetic acid (EDTA), and0.05M K₂ HPO₄, pH 7.3; and PBS-Tween containing 0.15M NaCl, 10 mM PC₄,and 0.05% Tween 20 (Fisher Scientific, Fair Lawn, NJ), pH 7.2.

Purified complement components

Partially purified C5, C6, C7, and C8 in addition to highly purified C9were isolated in accordance with the methods of Hammer et al. J. Biol.Chem. 256:3995, 1981. Prior to use, C9 was further purified by elutionfrom a hydroxylapatite column with a phosphate buffer gradient asdescribed by Biesecker and Muller-Eberhard, J. Immunol. 124:1291, 1980.[¹²⁵ I]Na (Amersham, Arlington Heights, IL) was used to radiolabel theC9 with the use of iodobeads (Pierce Chemical Co., Rockford, IL).

Polymerization of C9

Poly C9 was isolated by modification of the previously published methodof Podack and Tschopp, J. Biol. Chem. 257:15204 (1982). Five milligramsof purified C9 at 2 mg/ml, with a trace of ¹²⁵ I-C9 added, was incubatedat 37° C. for 63 hr in 10 mM Tris, 15 mM NaCl buffer containing 0.02%sodium azide, 25 μM p-nitrophenyl-p-guanidinobenzoate HCl, and 50 μg/mlof soybean trypsin inhibitor. The product was chromatographed on aBiogel A5M (Biorad, Richmond, VA) column (90×1.5 cm), and the ¹²⁵I-C9-containing peak eluting just behind the void volume was pooled.Electrophoresis in a 2.5 to 10% SDS-PAGE slab gel revealed the presenceof SDS non-dissociable poly C9 migrating as a broad band in the 2.5%portion of the gel (Podack and Tschopp Proc. Natl. Acad. Sci. USA79:574, 1982).

Purification of SC5b-9

SC5b-9 was isolated by the method of Ware et al. Molec. Immunol. 18:521(1981) modified as follows. One hundred milliliters of fresh human serumcontaining a trace amount of ¹²⁵ I-C9 was activated with zymosan, at 15mg/ml, for 2 hr at 37° C. The zymosan was removed by centrifugation, andpolyethylene glycol, 8% wt/vol, was added to the supernatant. Themixture was stirred at 4° C. for 30 min. The resulting precipitate wascollected, resuspended in lysine Sepharose buffer, and applied to ananti-C5 immunosorbent column with a bed volume of 40 ml. The column waswashed with 4 volumes of lysine Sepharose buffer and then eluted with 4Mguanidine HCl. The eluted protein was dialyzed against lysine Sepharosebuffer, concentrated, and applied to a Sepharose 6B-C1 column with a bedvolume of 500 ml. Two protein peaks eluting just behind the void volumewere pooled separately and concentrated. The final protein concentrationwas assessed by OD at 280 nm using an extinction coefficient of 1.0mg/ml-cm. Evaluation of these two pools by double diffusion andimmunoelectrophoresis showed the presence of C5, C6, C7, C8, and C9 ineach. Analysis by non-reduced 6.5% SDS-PAGE showed bands of M_(r)(relative molecular weight) consistant with C5, C6, C7, and C8.sup.αγchains; C8.sup.β chain; C9; and S protein, with minimal contamination byother proteins. SC5b-9 from both pools was used in subsequent tests.

Purification of MC5b-9

MC5b-9 was isolated by procedure of Ware et al. Molec. Immunol. 18:521(1981) modified as follows. Thirty milliliters of washed rabbiterythrocytes at 1.8×10⁹ cells/ml were incubated for 10 min at 37° C.with 50% fresh human serum (to which was added a trace amount of ¹²⁵I-C9) in GVBS++. Ghosts were sedimented at 31,000×g for 20 min, washedin 5 mM EDTA, resedimented, and then solubilized for 30 min in 5 mMsodium borate, 10 mM EDTA, and 1% SB-12 (Zwittergent 3-12, CalbiochemBehring Corp., La Jolla, CA) at room temperature (about 22° C. to 25°C.). The detergent-insoluble residue was removed by centrifugation, andthe detergent lysate was applied to a Biogel A5M column (90×1.5 cm). Thecolumn was eluted with 5 mM sodium borate and 50 mM NaCl buffer, pH 8.8,containing 0.02 % SB14 (Zwittergent 3-14, Calbiochem Behring Corp., LaJolla, CA). The trace radiolabeled C9 eluted as a single heavy molecularweight peak just behind the void volume. Nonreduced 7.5% SDS-PAGErevealed 7 bands with M_(r) consistent with C5, C6, C7, C8 C8, C9, andC9 dimer.

Preparation of anti-C9 neoantigen

A rabbit was immunized with purified poly C9, at 50 μg per week, incomplete Freund's adjuvant and bled weekly after the 4th week.

Absorption of anti-C9 neoantigen

Pooled normal human serum, purified C9, and partially purified pooledC5, C6, C7, and C8 were each linked to Sepharose 4B by the cyanogenbromide method, as outlined in the product instructions (Pharmacia FineChemicals, Piscataway, NJ). Antiserum to C9 neoantigen was seriallyabsorbed against each type of immunosorbent until negligible reactivityagainst monomeric C9 or human plasma remained, as assessed by the ELISAdescribed below.

Characterization of anti-C9 neoantigen

Antiserum to C9 neoantigen was initially characterized by doublediffusion in 1% agarose. Further characterization of specificity wasperformed by ELISA. Polystyrene 96-well plates (Dynatech Immulon II,Dynatech Lab Inc., Alexandria, VA) were coated for more than 24 hr at 4°C. with 100 μl/well of either poly C9 at 0.7 μg/ml, native C9 at 0.7μg/ml, or normal human plasma diluted 1:10, 1:100, or 1:1000 in a 0.05Msodium bicarbonate buffer, pH 9.6. The plates were washed 4 times withPBS-Tween and incubated 2 hr at room temperature with anti-C9 neoantigendiluted 1:2000 in PBS-Tween. Control wells were incubated with PBS-Tweenalone at this step. The plates were washed 6 times, and all of the cellswere incubated with goat anti-rabbit IgG alkaline phosphatase conjugate(Tago Inc., Burlingame, CA) at a 1:3000 dilution in PBS-Tween for 2 hr.Following this incubation, the plates were washed 6 times and developedwith p-nitrophenyl phosphate (Sigma, St. Louis, MO) dissolved in 10%diethanolamine buffer, pH 9.6, at 1 mg/ml. The color reaction wasstopped with 3M sodium hydroxide at 30 min, and the optical density (OD)at 405 nm was read in a MR 580 Dynatech Micro Elisa reader (Dynatech LabInc., Alexandria, VA). The net OD for each antigen was computed bysubtracting the values of the control well from the values of the testwell. Positive controls using polyclonal goat anti-C9 confirmed thebinding of C9 to the plates.

Purification of IgG

Rabbit anti-C9 neoantigen IgG was purified by DEAE-Affigel Bluechromatography (BioRad, Richmond, CA), using the protocol recommended bythe manufacturer.

Quantitative ELISA for SC5b-9

Dynatech Immulon II 96-well plates were coated with 100 μl of anti-C9neoantigen IgG at 5 μg/ml in 0.05M sodium bicarbonate buffer, pH 9.6,for more than 24 hr at 4° C. The plates were washed 4 times withPBS-Tween, and six wells for each sample were incubated for 3 hr with100 μl each of standard dilutions of SC5b-9 in PBS tween. The plateswere washed 6 times and three wells for each sample were incubated with100 μl of goat anti-C9 (Miles Laboratories, Naperville, IL), diluted1:2000 in PBS-Tween. At this step, three control wells for each samplewere incubated with PBS-Tween alone. AFter 2 hr, the plates were washed6 times, and all wells were incubated for an additonal 2 hr with 100 μlof swine anti-goat IgG alkaline phosphatase conjugate (Tago), diluted1:3000 in PBS-Tween. After a final eight washes, the plates weredeveloped with 200 μl/well of p-nitrophenyl phosphate, at 1 mg/ml, in10% diethanolamine buffer, pH 9.6 (FIG. 2). The reaction was stopped at45 min by adding 50 μl of 3M sodium hydroxide to each well, and OD 405was read in a Dynatech Micro Elisa reader. The net OD for each samplewas calculated by subtracting the average of triplicate control wellsfrom the average of triplicate wells that received goat anti-native C9.A standard curve was constructed by plotting net OD vs. concentration ofpurified SC5b-9. The assay was also run using purified MC5b-9 andpurified poly C9 as well as dilutions of normal plasma.

Characterization of anti-C9 neoantigen

By double diffusion, unabsorbed antiserum showed strong precipitin linesagainst poly C9 and MC5b-9. A line of partial identity was also seenwith native C9 (FIG. 3). After absorption with human serum Sepharose,double diffusion revealed single precipitin lines against poly C9 andMC5b-9, but no precipitin lines against plasma or native C9. After atotal of 18 immunosorbtion steps with normal serum Sepharose, C9Sepharose, and C5, C6, C7, and C8 Sepharose, antiserum specificity wastested in a more sensitive fashion by ELISA. ELISA data supported thespecificity of the antiserum for C9 neoantigen as shown in Table Iindicating that antibodies to native C9 determinants as well as to otherplasma proteins were removed.

                  TABLE I                                                         ______________________________________                                        ANTISERUM SPECIFICITY BY ELISA                                                Antigen        Net OD.sup.a                                                   ______________________________________                                        Native C9      0.011                                                          Poly C9        0.650                                                          Plasma 1:10    0.008                                                          Plasma 1:100   -0.048                                                         Plasma 1:1000  0.001                                                          ______________________________________                                         .sup.a Mean net OD 405 from triplicate wells read at 30 min. Native C9 an     poly C9 were both plated at 0.7 μg/ml. AntiC9 neoantigen was applied t     plates at 1:2000 dilution. The second antibody was a goat antirabbit          alkaline phosphatase conjugat e at a dilution of 1:3000.                 

Quantitative ELISA for SC5b-9

In the quantitative ELISA, purified SC5b-9 showed a dose-responserelationship with OD increasing with increasing amounts of antigen (FIG.4). In other tests, the quantitative ELISA was shown to be sensitive toas little as 100 ng of SC5b-9/ml (FIG. 5). In order to show that theELISA was detecting C9 neoantigen, standard curves were run withpurified poly C9, MC5b-9, and dilutions of normal human plasma. Asexpected, both poly C9 and MC5b-9 showed dose-response relationships,with the OD increasing in response to the increasing amounts of antigen(FIG. 6). Similar curves were obtained when SC5b-9, poly C9, and MC5b-9were diluted in 2% normal human plasma. Dilutions of normal human plasmafailed to show a response in the quantitative ELISA, showing that theassay did not detect proteins present in normal plasma (FIG. 7).

The results presented above demonstrate an easily performed, sensitiveELISA which can detect and quantitate fluid phase C5b-9 complexes. Inthis assay, antibodies highly specific for the C9 neoantigen were usedin a sandwich fashion with antibodies directed to native C9 epitopes.C5b-9 complexes in solution are bound immunologically by rabbit anti-C9neoantigen IgG coated on a plate. Since C9 will bind to the plate onlyif the C9 neoantigen is expressed as in an assembled C5b-9 complex, thebinding of the goat anti-native C9 can then be used to quantitate theamount of C5b-9 complexes bound on the plate, which is in turnproportional to the amount of C5b-9 complexes present in the testsolution. By comparison to a standard curve generated with purifiedSC5b-9, the amount of SC5b-9 in an unknown sample can be assessed.

Of course, the ELISA of the present invention can be constructed usingeither monoclonal or polyclonal antibodies directed against any twoepitopes that are found in assembled C5b-9 complexes. For instance, amonoclonal antibody to the C9 neoantigen and a biotinylated monoclonalto native C9 can be profitably employed with an avidin labelled enzymewhich produces a color reaction upon addition of a suitable substrate.Alternatively anti-C5 could be used to coat the plates as a captureantibody and anti-native C9 then used as the developing antibody. Aproblem arises in this second alternative with the potential of theuncomplexed C5 in the sample to compete with SC5b-9 for binding to thecapture antibody. This problem is avoided in the assays by the use of acapture antibody specific for a neoantigen present only in the assembledC5b-9 complex. Likewise, the enzyme employed for producing the colorreaction may be preferably either alkaline-phosphatase or horseradishperoxidase or the like.

In summary, the major advantages of the present ELISA over previouslypublished assays are that it is more sensitive, it gives a directquantitation of SC5b-9 in nanograms per milliliter, and it can beperformed without the use of radioactive materials, provided the SC5b-9used for the standard curve is purified without the use of tracelabeling. The sensitivity of the present assay, about 100 ng ofSC5b-9/ml, is such that as little as a 2% activation of the terminalcomplement components in normal spinal fluid can be detected based onthe published concentration of C9 in normal spinal fluid and thestoichiometry of SC5b-9 (Kolb and Muller-Eberhard, 1975; Morgan et al.,1984).

It should be noted that the optimal dilutions of clinical specimen inthe present assay is found to be 1:50 for plasma samples and 1:2 to 1:10for cerebrospinal fluid samples. In this range, most of the samplevalues fall on the steep, approximately linear portion of the curve, andinterference with the assay by other proteins in the sample is minimal.

The present invention now makes it possible to use an ELISA toquantitate activation of the terminal pathway of the complement cascade.The assay measures an actual product of activation rather thanconsumption of components and can, therefore, be used to detect andquantitate activation of the terminal complement cascade incircumstances where normal levels of components may not be known or maybe altered by other factors, such as an acute phase reactant response ora leaky blood-compartment barrier. It has been found that the presentquantitative ELISA can measure SC5b-9 in the spinal fluid of patientswith a variety of neurologic diseases. The sensitvity and specificity ofthe present assay makes it possible to monitor terminal complementactivation in a variety of human diseases where such activation may beinvolved.

A kit in accordance with the present invention comprises containerscontaining a plurality of different antibodies (first, second and thirdantibodies as described herein), enzyme substrate, SC5b-9 standard,microtiter plate, instructions for performing the assay and the likeincluding other accessories routinely found in similar kits.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application and thescope of the appended claims.

We claim:
 1. A method for assaying activation of terminal complementcascade, comprising in sequence the steps of:(a) coating a suitablesurface with a sufficient amount of a first antibody having specificityfor poly C9 neoantigen; (b) washing said surface with a suitable mediumto remove unbound antibody; (c) incubating said surface with a sample tobe assayed for sufficient time so that binding reaction between saidsample and the first antibody is substantially complete; (d) washingsaid surface with a suitable medium to remove unbound sample; (e)incubating said surface with a second antibody different from the firstantibody and specific for a constituent of the terminal cascade forsufficient time so that binding between antigen and the second antibodyis substantially complete; (f) thereafter washing the surface with asuitable medium to remove unbound second antibody; (g) incubating saidsurface with a third antibody which recognizes the second antibody andwhich is conjuncted with a suitable enzyme for a sufficient time so thatthe binding between said second and third antibodies is substantiallycomplete; (h) washing said surface with a suitable medium to removeunbound third antibody; (i) incubating the surface with a substratespecific for the conjugated enzyme for sufficient time so that theenzyme-substrate reaction is substantially complete; and (j) measuringthe product resulting from enzyme-substrate reaction relative to knownamounts of SC5B-9 standard similarly treated as in steps (a) thru (j).2. The method of claim 1 wherein said first, second or third antibody ismonoclonal or polyclonal.
 3. The method of claim 2 wherein said enzymeis alkaline phosphatase or horseradish peroxidase.
 4. A method forassaying activation of terminal complement cascade, comprising insequence the steps of:(a) coating a suitable surface with a sufficientamount of a first antibody having specificity for poly C9 neoantigen;(b) washing said surface with a suitable medium to remove unboundantibody; (c) incubating said surface with a sample to be assayed forsufficient time so that binding reaction between said sample and thefirst antibody is substantially complete; (d) washing said surface witha suitable medium to remove unbound sample; (e) incubating said surfacewith a second antibody different from the first antibody and specificfor a constituent of the terminal cascade for sufficient time so thatbinding between antigen and the second antibody is substantiallycomplete, said second antibody being conjugated with a first ligandwhich binds a second ligand on an enzyme applied subsequently in step(g); (f) thereafter washing the surface with a suitable medium to removeunbound second antibody; (g) incubating said surface with aligand-conjugated enzyme which binds the ligand on the second antibodyfor a sufficient time so that the binding between said second antibodyand enzyme is substantially complete; (h) washing said surface with asuitable medium to remove unbound enzyme; (i) incubating the surfacewith a substrate specific for said enzyme for sufficiet time so that theenzyme-substrate reaction is substantially complete; and (j) measuringthe product resulting from enzyme-substrate reaction relative to knownamounts of SC5b-9 standard similarly treated as in steps (a) thru (j).5. The method of claim 4 wherein said first ligand is biotin and thesecond ligand is avidin.
 6. The method of claim 5 wherein said enzyme isalkaline phosphatase or horse-radish peroxidase.